Cloning ‐free template DNA preparation for cell‐free protein synthesis via two‐step PCR using versatile primer designs with short 3′‐UTR

Cell‐free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time‐consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two‐step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3′‐untranslated region (3′‐UTR) sequence that facilitates translation. Application of the short 3′‐UTR to two‐step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N‐ or C‐terminal tags, and truncated proteins. Our method supports the cloning‐free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS.

Here, we developed a rapid and efficient two‐step PCR method to construct linear DNA templates for a wheat germ cell‐free protein synthesis system. We characterized an effective short 3′‐UTR sequence that facilitates translation. Application of the short 3′‐UTR to two‐step PCR enabled the generation of various transcription templates without cloning procedures.

Source: Genes to Cells - December 1, 2017 Category: Genetics & Stem Cells Authors: Mika Nomoto, Yasuomi Tada Tags: BRIEF REPORT Source Type: research